Physical and functional probing of DEAD-box proteins as general RNA chaperones

描述(由申请人提供)：结构化RNA执行的几乎每个过程都需要DExD/H-box蛋白，从前mRNA剪接和翻译到蛋白质和RNA的细胞内运输。这些蛋白质被认为利用来自ATP的能量来促进RNA构象变化和折叠过渡，但在分子水平上对它们如何操纵RNA结构知之甚少。虽然DExD/H-box蛋白的许多RNA和RNA-蛋白质靶标都很大、很复杂，而且很难在体外进行研究，但2002年的研究表明，粗脉孢子菌Cyt-19蛋白参与了几个线粒体I组内含子的折叠。进一步的工作表明，细胞色素T-19也能与第二组内含子相互作用，这表明它具有一般的RNA伴侣活性，相关的酵母蛋白Mss 116p的功能也被证明是相似的。本项目的目标是使用定义明确、易于处理的细胞色素T-19和I组RNA系统来剖析DExD/H-box蛋白对RNA伴侣活性的作用机制。除了增加对基本细胞过程的了解外，这项工作对疾病也有影响，因为包括丙型肝炎病毒在内的病毒的复制需要DExD/H-box蛋白，而人类DExD/H-box蛋白的过度表达与结肠癌和前列腺癌有关。在当前供资期间取得的进展导致了RNA伴侣活动的一般模式，本提案围绕该模式展开。首先，Cyt-19可以不分青红皂白地破坏RNA结构，展开非同源I组内含子的自然状态和长期错误折叠的构象，其效率取决于RNA物种的相对稳定性，而不是任何特定的结构特征。然而，它的活性并不是完全不分青红皂白的；它通过形成额外的“系留”相互作用优先作用于结构RNA，并且只有当P1不与RNA的其余部分“对接”进入第三级接触时，它才会解开I组内含子(P1)的一个短螺旋。这些结果表明，细胞色素T-19可能会破坏不能正确包装的优先错误折叠的RNA。目前的建议的目标是进一步深入研究DExD/H-box蛋白对RNA伴侣活性的物理基础，利用细胞色素T-19及其生理底物探索特异性，并测试关于三级接触系留和抑制的机制和意义的假说。具体目的是：1)使用寡核苷酸置换试验和定向羟基自由基足迹法来探测I组内含子上的RNA伴侣活性的途径；2)确定CyT-19是否以非同源RNA所不具有的特异性作用于其同源I组内含子；3)进一步探讨三级接触形成的抑制作用，包括通过确定这种抑制是否存在于不同的结构元件和不同的I组内含子中来检验该抑制是伴侣活性的一般特征的假说；4)验证以下假设，即这种拴系相互作用是由细胞色素T-19和相关的DExD/H-box蛋白的一个高度碱性的‘尾’形成的，并且这种相互作用在局部解离过程中可以保持不变。这些结果有望在RNA代谢的各个方面指导DExD/H-box蛋白的模型。 与公共卫生相关：该项目的目标是了解RNA伴侣蛋白在RNA折叠到特定结构以及结构之间的交换时如何帮助RNA。这些蛋白质是病毒复制所必需的，并与人类癌症有关，因此了解它们的功能对于理解和最终治疗人类疾病非常重要。

DESCRIPTION (provided by applicant): DExD/H-box proteins are required for virtually every process carried out by structured RNAs, from pre-mRNA splicing and translation to intracellular trafficking of proteins and RNAs. These proteins are thought to use energy from ATP to facilitate RNA conformational changes and folding transitions, but relatively little is known on a molecular level about how they manipulate RNA structure. While many of the RNA and RNA-protein targets of DExD/H-box proteins are large, complex, and difficult to study in vitro, it was shown in 2002 that the Neurospora crassa CYT-19 protein functions in folding of several mitochondrial group I introns. Further work indicated that CYT-19 can also interact productively with group II introns, indicating that it possesses general RNA chaperone activity, and the related yeast protein Mss116p was shown to function similarly. The goal of this project has been to use the well-defined, tractable system of CYT-19 and group I RNAs to dissect the mechanisms of RNA chaperone activity by DExD/H-box proteins. In addition to increasing knowledge of essential cellular processes, this work has implications for diseases, as DExD/H-box proteins are required for replication of viruses including HCV, and overexpression of human DExD/H-box proteins is linked to colon and prostate cancer. Progress during the current funding period led to a general model for RNA chaperone activity, around which this proposal is centered. First, CYT-19 can disrupt RNA structure indiscriminately, unfolding both the native state and a long-lived misfolded conformer of a non-cognate group I intron with efficiencies that depend on the relative stabilities of the RNA species but not on any specific structural features. However, its activity is not fully indiscriminate; it acts preferentially on structured RNAs by forming an additional 'tethering' interaction, and it unwinds a short helix of a group I intron (P1) only when P1 does not 'dock' into tertiary contacts with the rest of the RNA. These results suggest that CYT-19 may disrupt preferentially misfolded RNAs that cannot pack correctly. The goals of the current proposal are to delve further into the physical basis of RNA chaperone activity by DExD/H-box proteins, to probe for specificity using CYT-19 and its physiological substrates, and to test hypotheses on the mechanisms and implications of tethering and inhibition by tertiary contacts. Specific Aims are: 1) to use an oligonucleotide displacement assay and directed hydroxyl radical footprinting to probe pathways of RNA chaperone activity on group I introns; 2) to determine whether CYT-19 acts on its cognate group I introns with specificity that is absent with a non-cognate RNA; 3) to probe further the inhibition by tertiary contact formation, including testing the hypothesis that the inhibition is a general feature of chaperone activity by determining whether it is present for different structural elements and within a different group I intron; and 4) to test the hypotheses that the tethering interaction is formed by a highly basic 'tail' of CYT-19 and related DExD/H-box proteins, and that this interaction can remain intact during local unwinding. Results are expected to guide models for DExD/H-box proteins in all aspects of RNA metabolism. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand how RNA chaperone proteins assist RNAs as they fold to specific structures and exchange between structures. These proteins are required for viral replication and are linked to human cancer, so understanding how they function is important for understanding and ultimately treating human disease.